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Cardiovascular Research 1999 41(3):620-628; doi:10.1016/S0008-6363(98)00281-8
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Copyright © 1999, European Society of Cardiology

Inositol-1,4,5-trisphosphate increases contractions but not L-type calcium current in guinea pig ventricular myocytes

Tomoaki Saeki1,1, Jian-Bing Shen and Achilles J. Pappano*

Department of Pharmacology, University of Connecticut Health Center, Farmington, CT 06030, USA

* Corresponding author. Tel.: +860-679-2410; fax: +860-679-3693; e-mail: pappano@nsol.uchc.edu

Received 12 May 1998; accepted 21 August 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: We studied the effects of intracellularly applied inositol-1,4,5-trisphosphate (InsP3) to test the hypothesis that InsP3 is a messenger for stimulation of L-type calcium current (ICa(L)) and contractions by muscarinic agonists. Methods: Voltage clamp pulses elicited ICa(L) that evoked contractions recorded with an edge detector in single guinea pig ventricular myocytes superfused with Tyrode’s solution (36°C). InsP3 or cyclic AMP (cAMP) was dialyzed into the cell at selected times via the patch electrode. Results: InsP3 (1–10 µM) transiently increased isotonic contractions when applied for 4–5 min; higher concentrations (50–300 µM) caused a sustained decrease in contractions. InsP3 had no effect on ICa(L) at any concentration tested. Caffeine (10 mM)-induced contractures were increased and decreased, respectively, at 3 and 100 µM InsP3. Pentosan polysulfate (50 µg/ml), an InsP3 receptor antagonist, opposed the increased contractions by InsP3. Intrapipette cyclic AMP (10–300 µM) caused sustained increases of ICa(L) and contractions. Cyclic AMP, but not InsP3, also increased ICa(L) when intrapipette Cs+ suppressed K+ currents. Conclusions: Increased myocyte shortening at low InsP3 concentrations accords with receptor-initiated sarcoplasmic reticulum Ca2+ release. The transient stimulation of contractions at low concentrations and the sustained reduction of contractions at high concentrations are not consistent with a role for InsP3 in the persistent increase of contractions by muscarinic agonist in ventricular muscle and myocytes. The failure of InsP3 to change ICa(L) when contractions were increased or decreased militates against the L-type calcium channel being an effector of InsP3.

KEYWORDS Guinea pig; Ventricular myocytes; Cell contractions; Sarcoplasmic reticulum; InsP3; Excitation–contraction coupling; Intracellular dialysis; L-type Ca2+ current; Cyclic AMP


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Signalling by muscarinic receptors (mAChR) responding to the parasympathetic neurotransmitter, acetylcholine (ACh), includes stimulation as well as inhibition of the heartbeat (reviewed in [1]). Muscarinic agonists increase contraction force in papillary muscle [2–5]and in myocytes isolated from guinea pig heart [6, 7]. Muscarinic agonists also increase the synthesis of inositol-1,4,5-trisphosphate (InsP3) in guinea pig [3, 8]and rat [9]ventricular myocytes. A causal relationship has been suggested between the increased level of InsP3 in cardiac tissues and the positive inotropic effect of muscarinic agonists, ACh and carbachol (CCh). Thus, CCh caused a sustained increase of InsP3 content that preceded the sustained increase of contractions in guinea pig atria [3]. The concentration dependence for the former effect is the same as for the latter [3, 9, 10]. InsP3 released Ca2+ from the sarcoplasmic reticulum (SR) in mechanically skinned [11]and chemically permeabilized heart tissue [12–14]and in voltage-clamped ventricular myocytes [15]. While the rate of Ca2+ release was slower with InsP3 than with calcium-induced calcium release (CICR) [11, 16], these results do not preclude InsP3 from functioning as a Ca2+-mobilizing messenger for neurotransmitters like ACh.

More recently, Gallo et al. [8]reported that CCh increased the L-type Ca2+current (ICa(L)) in guinea pig ventricular myocytes. This action of CCh occurred at concentrations that also increased InsP3 content and it was proposed that this messenger was responsible for the CCh effect on ICa(L). Subsequently, others reported that CCh increased the intracellular Ca2+ transient in rat ventricular myocytes [17]; it was assumed that the CCh effect arose from a larger Ca2+ trigger, namely, ICa(L). While our results agreed with the view that CCh increased intracellular Ca2+ transients and contractions, we concluded that the mechanism involved an increase of SR Ca2+ content [6, 7]. Moreover, CCh did not change ICa(L) when it augmented intracellular Ca2+ transients and contractions in guinea pig ventricular myocytes [6, 7]. However, there is no direct test of the InsP3 hypothesis for activation of ICa(L). This messenger could act on the cardiac L-type Ca2+ channel to increase its availability or conductance as reported in T-tubular membranes of skeletal muscle [18]. According to the Ca2+-mobilizing action of InsP3, this messenger might act indirectly via the SR to produce increases not only of contraction but also of ICa(L). Neither InsP3 nor ACh stimulated ICa(L) in guinea pig ventricular myocytes [19]. In these experiments, the pipette was filled with ‘minimum intracellular solution’ (CsCl, 80 mM; CsOH, 40 mM; MgCl2, 2 mM; EGTA, 10 mM and HEPES, 10 mM; pH=7.4) lacking ATP and cyclic AMP and to which non-hydrolyzable guanine nucleotides were added. The lack of effect of ACh or InsP3 on ICa(L) might have resulted from the experimental conditions.

The present experiments test the hypotheses that InsP3 increases ICa(L) and that it serves as a second messenger for muscarinic agonist-induced contractile stimulation. Because the InsP3 effect on trigger Ca2+ entry through L-type Ca2+ channels was reported in cells dialyzed with Cs+ and EGTA [8], InsP3 could act on a channel-related protein to increase ICa(L). The effect of the continuous application of intracellular InsP3 on excitation–contraction coupling is not known. Therefore, experiments were also done with a K+-rich pipette solution that allowed evaluation of InsP3 effects on ICa(L) and on cell contractions. Additionally, we compared the effects of InsP3 with those of cAMP, each of which was sequentially added to the pipette-filling solution. A preliminary account of these findings has been reported [20].


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell isolation
Single ventricular myocytes were enzymatically isolated from the hearts of male and female guinea pigs (250–350 g) that had been anesthetized with sodium pentobarbital (30 mg/kg, intraperitoneally) and anticoagulated with heparin (400 IU, i.p.). The investigation conforms with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). The heart was retrogradely perfused with Tyrode’s solution for 5 min at a rate of 8–10 ml/min through an aortic cannula in a Langendorff apparatus. The composition of Tyrode’s solution was (in mM): NaCl 135, KCl 5.4, CaCl2 1.8, MgCl2 1.0, NaH2PO4 0.33, HEPES 10, glucose 27.5, and the pH was adjusted to 7.4 with NaOH. Next, Ca-free Tyrode’s solution was perfused for 45 to 60 s until the heart stopped beating. Subsequently, the heart was perfused with Tyrode’s solution containing 30–40 µM Ca and containing 36 mg of collagenase (Boehringer Manheim, type A) and 3.6 mg of protease (Sigma, type IV) in 50 ml. After recirculation for 10 min, the enzymes were washed out by perfusion with 50 ml of recovery solution. Recovery solution contained (in mM): potassium aspartate 130, K2ATP 5, HEPES 5, glucose 20, and the pH was adjusted to 7.4 with KOH. The ventricles were removed and the cells were dispersed in recovery solution and kept at 4°C for at least 1 h. An aliquot of cell suspension was placed in a recording chamber (500 µl volume) that was mounted on the stage of an inverted microscope. After 10 min, it was superfused with Tyrode’s solution (2 ml/min); the glucose concentration was reduced to 10 mM to perform experiments. The temperature was 36±0.5°C unless otherwise indicated.

2.2 Electrophysiology
An EPC 7 patch clamp amplifier (List Electronics, Germany) was used to deliver voltage clamp pulses in whole cell mode. All voltage commands and current data acquisition were controlled by an IBM-compatible computer equipped with pClamp software (version 5.5, Axon Instruments, Burlingame, CA, USA) and a Labmaster TL-1 interface (Axon Instruments). Electrodes were prepared from glass capillaries (1.1 mm, i.d.; 1.3 mm o.d.) and filled with a pipette solution whose composition was (in mM): potassium aspartate 120, KCl 30, Na2ATP 4, MgCl2 1.0 and HEPES 5, pH=7.2 (with KOH). The resistance was 2 to 4 M{Omega}. In some experiments, the pipette was filled with a Cs+-rich solution containing EGTA, to replicate the conditions used by others [8]and to test the possibility that InsP3 could act on a channel-related protein to increase ICa(L). This solution was composed of (in mM): cesium aspartate 135, NaCl 10, MgATP 5, EGTA 5 and HEPES 10; pH=7.3 (with CsOH). The pipette was connected to the amplifier by a Ag–AgCl wire, and the tip was gently pushed against the cell surface. Negative pressure was applied to the pipette interior until a gigaohm seal was formed. After the electrode capacitance was compensated electronically in the cell-attached mode, the cell membrane was ruptured by additional negative pressure. The voltage clamp protocol consisted of a 1-s ramp depolarization from –80 to –30 mV followed 300 ms later by a voltage jump to 10 mV for 300 ms and then sequential repolarizing steps to –30 and –80 mV. The protocol was applied every 5 s. The ramp depolarization was used to avoid ‘escape’ of voltage control, which can occur upon a voltage jump from –80 to –30 mV. The abbreviated action potentials during ‘escape’ can cause contractions by releasing Ca2+ from the SR and thereby change the Ca2+ content of SR for the subsequent voltage jump that activates ICa(L).

Net inward current during voltage jumps to +10 mV was used to estimate ICa(L). These estimates are less complicated when outward K+ currents are suppressed by Cs+ in the bath- and pipette-filling solutions. Superposition of membrane currents revealed no change in charge movement during test pulses to +10 mV when InsP3 was applied in the presence of Cs+-rich pipette solution or in the presence of K+-rich pipette solution (Fig. 2). In two experiments with K+-rich pipette solution, which allows contraction measurements, there was no difference in Cd2+-sensitive current, taken as ICa(L), in InsP3 (see Fig. 2). By contrast, cAMP increased ICa(L) in either K+-rich or Cs+-rich pipette solution. Therefore, the lack of change in net inward membrane current in InsP3 indicates that InsP3 did not change charge movement during ICa(L).


Figure 2
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  		Fig. 2 Low concentrations of InsP3 increase contractions but not ICa(L). The voltage clamp protocol is as in Fig. 1. (A) ICa(L) and contractions in control and at 1 and 4 min after the addition of 5 µM InsP3. The right-most panel shows membrane current and contraction records from control ({square}), with that at 1 min in InsP3 ({blacksquare}) superimposed. (B) Recordings from another myocyte (22°C) illustrating the Cd2+-sensitive membrane current and associated contractions during 100 ms voltage jumps from –30 to +10 mV in control and at 1 and 6 min after the addition of 3 µM InsP3. The panel on the right shows membrane current from control ({circ}) and at 1 min in InsP3 (bullet). Calibrations for current and cell shortening are indicated; zero current is marked by a thin horizontal line to the left of the control records in (A) and (B).

 
2.3 Cell contraction
With the cell shortening along its long axis, displacement of one end of the cell edge was used to indicate the extent of cell contraction. A video-edge detector system (Crescent Electronics, Sandy, UT, USA) tracked cell edge motion. A microscope-magnified (400x) cell image was observed continuously on a high-resolution black-and-white TV monitor via a sequential scanning video camera attached to a sideport of the microscope. The camera position was rotated so that the video monitor raster lines were parallel with the long axis of the cell. The video dimension analyzer monitored a selected raster line for light intensity differences between the end of the myocyte and the surrounding field. The temporal resolution of this detector was 16.7 ms and motion of as little as 0.02 µm could be detected. The signal from the detector was sent to a strip chart recorder and to a videocassette recorder for storage and off-line analysis.

2.4 Cell dialysis and rapid superfusion
The method for intracellular application of InsP3 and cAMP through the pipette solution was the same as that used previously in our laboratory [15]. In experiments with externally applied caffeine, the alkaloid was superfused over the cell surface by a solenoid-controlled device that changed the solution composition in <1 s [6].

2.5 Data analysis
Experimental results are expressed as means±SEM. Comparisons between means were made with Student’s t-test with p≤0.05 taken as being statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 InsP3 experiments with K+-rich pipette solution
3.1.1 Concentrations ≥50 µM
Typical effects of a relatively high (100 µM) InsP3 concentration are shown in Fig. 1. A voltage jump from –30 to +10 mV for 300 ms evoked an ICa(L) of 0.7 nA (estimated as net inward current) and a contraction of 9 µm during the control period. Thereafter, the pipette was filled with a solution to which 100 µM InsP3 had been added. At 3 min after the introduction of InsP3, ICa(L) was unchanged, at 0.7 nA, but cell shortening had diminished by 33%, to 6 µm. Myocyte contractions returned to the initial value (9 µm) after removing InsP3 from the pipette-filling solution while net inward ICa(L) remained constant at 0.7 nA. Subsequent addition of cAMP (300 µM) to the pipette solution caused ICa(L) and cell contractions to increase in parallel. A transient inward (TI) current was evident upon repolarization to –30 mV in 300 µM cAMP. Net ICa(L) increased to 3.1 nA and cell shortening was 29 µm in cAMP whose effects were reversed by removal from pipette solution (Fig. 1, right-hand panel). There was some run-down of contractions; at the time of washout, cell shortening was 4 µm.


Figure 1
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  		Fig. 1 Effects of high concentrations of InsP3 (100 µM) and cAMP (300 µM) on ICa(L) and contractions in a guinea pig ventricular myocyte. The voltage clamp protocol consisted of a 1-s ramp depolarization from –80 to –30 mV followed 300 ms later by a voltage jump to 10 mV for 300 ms and then sequential repolarizing steps to –30 and –80 mV. The protocol was applied every 5 s. Membrane current is shown in the upper trace and cell contraction is shown in the lower trace in each panel; the panels are labelled according to the experimental conditions. From left to right, the records are control, at 3 min in InsP3, at 5 min after InsP3 washout, at 3 min in cAMP and at 7 min after cAMP washout. Calibrations for current, motion and time apply to all panels; zero current is indicated by the thin horizontal line at the left of the control current trace.

 
In three other cells tested with 50, 100 and 300 µM InsP3 in the pipette solution, contractions were reversibly decreased while ICa(L) remained unchanged.

3.1.2 Concentrations from 1 to 10 µM
At pipette concentrations from 1 to 10 µM, InsP3 evoked a biphasic effect, an increase followed by a decrease in cell contractions. Results from two experiments with 5 and 3 µM InsP3, respectively, are shown in Fig. 2. Before the addition of 5 µM InsP3 (Fig. 2A), ICa(L) was 1.3 nA and isotonic shortening was 5.9 µm. At 1 min after the introduction of InsP3, ICa(L) was 1.3 nA and cell contraction increased by 30% to 7.6 µm. At 4 min in InsP3, ICa(L) was 1.3 nA and cell shortening had returned to the control level of 5.9 µm. Superimposed records from control and at 1 min in InsP3 (Fig. 2A) revealed no change in either the peak or the inactivation of current at +10 mV when cell shortening had increased in InsP3. While the rate of shortening did not change, the duration of contraction at half-maximal amplitude (t0.5) increased from 200 to 220 ms. In another test (Fig. 2B), 0.3 mM Cd2+ was used to suppress the L-type Ca2+ current and thereby define the magnitude of this current in the absence and presence of InsP3. It has been concluded that 0.1 mM Cd2+ completely blocked ICa(L) in rabbit ventricular myocytes [21]. The records show the Cd2+-sensitive current in control and at 1 and 6 min after 3 µM InsP3. While cell shortening increased by 22%, from 5.6 to 6.9 µm at 1 min in InsP3, the contraction effect had dissipated by 6 min in InsP3. The t0.5 was 180 ms in control and did not change in InsP3. There was no appreciable difference in the Cd2+-sensitive current when InsP3 caused contractions to increase (Fig. 2B, right-hand panel). (There is an outward tail current upon repolarization to –30 mV; this current simply ran down during the time course of the experiment and was not affected by InsP3). Overall, t0.5 tended to increase from an average of 179±8.9 ms in control to 204±17.4 ms in 1–10 µM InsP3 (n=13); however, the increment of 25±17.5 ms did not attain statistical significance (p=0.18).

The concentration dependence for the effect of InsP3 on cell shortening is summarized in Fig. 3. The column labelled ‘0’ gives the extent of contraction in the control period and includes all of the cells to which InsP3 was eventually applied. When compared to the respective controls, InsP3 increased cell shortening significantly at 1 µM (p=0.02), 3 µM (p=0.001) and 10 µM (p=0.03). The increase in cell shortening was measured at 1–2 min because it was maximal during this interval in low concentrations of InsP3. Contractions in 50–300 µM InsP3 diminished significantly to 2.8±0.8 µm (p=0.02). The reduction in cell shortening at ≥50 µM InsP3 was evaluated at 3–4 min when it had attained steady-state. The L-type Ca2+ current was not significantly changed by InsP3 at either the low concentration range, where biphasic contraction effects occurred, or at the high concentration range, where cell shortening simply decreased.


Figure 3
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  		Fig. 3 Summary of the concentration-dependence of the effect of InsP3 on cell shortening. Cell shortening is given in µm; data at IP3=0 µM indicate the extent of cell shortening, which averaged 5.4±0.38 µm in n=21 cells in the absence of the phosphoinositide. The control values of shortening for each InsP3 concentration group are [µm±SEM]: 5.2±1.02 (1 µM), 5.6±0.44 (3 µM), 5.0±0.71 (10 µM) and 5.5±1.84 (50–300 µM). Numbers in parentheses are the cells tested at each concentration. *indicates p≤0.05.

 
The effects of InsP3 were also tested on contractures induced by rapid superfusion of 10 mM caffeine. We used the voltage clamp protocol of Fig. 1 for periods of 2–3 min and then applied caffeine within 5 s after electrical stimulation ceased. We confirmed the observation of others [12]that 3 µM InsP3 potentiated the contracture induced by rapid extracellular application of 10 mM caffeine (n=2; data not shown). At 100 µM InsP3, caffeine-induced contractures were decreased, as were contractions. Before dialysis with InsP3, contractions averaged 3.2±0.56 µm and contractures were 3.0±0.37 µm (n=6). Contractions diminished by 34% to 2.1±0.19 µm (p=0.04) after 3–6 min of dialysis with InsP3. The caffeine-induced contractures were likewise reduced during InsP3 dialysis by 53% to 1.4±0.39 µm (p=0.006). The suppressant effect of 100 µM InsP3 was reversed by dialysis with InsP3-free pipette solution in three of the cells tested. In the remainder, cells were dislodged or damaged before the washout period reached 3 min.

3.1.3 Experiments with pentosan polysulfate (PPS)
Heparin and related polysulfates act as antagonists of the InsP3 receptor. Pentosan polysulfate (50 µg/ml) was used to ascertain if it opposed the stimulatory effect of 3 µM InsP3 on contractions. An experiment of this type is shown in Fig. 4. The net ICa(L) and contraction in controls were 1.8 nA and 6.5 µm, respectively. There was no change in ICa(L) when PPS was added to the pipette solution. By contrast, cell shortening increased by 46% to 9.5 µm (Fig. 4). When InsP3 (3 µM) was added to the pipette solution containing PPS, there was no change in either ICa(L) (1.8 nA) or cell shortening (9.5 µm). Neither ICa(L) nor cell shortening changed when InsP3 was removed from the pipette solution. However, upon washout of PPS from the pipette solution, myocyte contractions decreased towards control values, while net ICa(L) remained at 1.8 nA. Similar results were obtained in two additional experiments where contractions increased by 20 and 47% in PPS (50 µg/ml) and InsP3 (3 µM) had no additional stimulatory effect in the presence of PPS. In these two experiments, 3 µM InsP3 alone caused contractions to increase by 20 and 33%, respectively.


Figure 4
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  		Fig. 4 Pentosan polysulfate (PPS, 50 µg/ml) prevents stimulation of contractions by InsP3 (3 µM). The voltage clamp protocol used was the same as in Fig. 1. From left to right, the records are of control, at 5 min in PPS, at 3 min after adding InsP3 on top of PPS, at 4 min after washout of InsP3, and at 5 min after PPS washout. Calibrations for current, motion and time apply to all panels; zero current is indicated by a thin horizontal line to the left of the control record.

 
3.2 InsP3 experiments with Cs+-rich pipette solution
3.2.1 Concentrations ≥50 µM
The original experiments in which InsP3 was proposed to increase ICa(L) were done with a Cs+-rich pipette solution [8]. We did not detect any effect of InsP3 on ICa(L); contractions were absent because of the presence of 5 mM EGTA in the pipette solution. The results from one of these experiments are shown in Fig. 5A. The net ICa(L) was 2.3 nA in the control (Fig. 5Aa); adding 50 µM InsP3 to the pipette solution did not change ICa(L) significantly (2.2 nA) during the 5 min test period (Fig. 5Ab). There was a small reduction of ICa(L), to ~1.7 nA, after removing InsP3 (Fig. 5Ac). Subsequent addition of 50 µM cAMP to the pipette solution caused ICa(L) to increase to 3.6 nA (Fig. 5Ad) and this effect was reversed by removal of cAMP (Fig. 5Ae). The results of three experiments with 50–100 µM InsP3 are shown in Fig. 5B. No significant change to ICa(L) was caused by InsP3 (p=0.19) when compared to control. A significant (p=0.04) decline of ICa(L) occurred after washout of InsP3, but the reason for this is unknown. The addition of cAMP (50 or 100 µM) increased ICa(L) as expected.


Figure 5
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  		Fig. 5 cAMP (50 µM) but not InsP3 (50 µM) increases ICa(L) in the presence of intrapipette Cs+ and EGTA (see Section 2). (A) Results from one cell showing current traces (a–e) above and the time course of the experiment in the graph below, with the times at which the individual records were taken being indicated. The voltage clamp protocol is the same as in Fig. 1. (B) Summary of three experiments performed with Cs+-rich pipette solution in which the effects of 50–100 µM cAMP were compared with those of 50–100 µM InsP3.

 
3.2.2 Concentrations from 2 to 10 µM
Additional experiments were performed with Cs+-rich pipette solution to examine the effects on ICa(L) of 2–10 µM InsP3. At these concentrations in K+-rich pipette solution, InsP3 transiently increased the extent of contractions. However, in the presence of Cs+-rich pipette solution, there was no change in ICa(L) at 2–10 µM InsP3, while cAMP displayed its usual stimulatory effect. An example typical of the results obtained in six such experiments is shown in Fig. 6. When the pipette solution was switched from Cs+/EGTA alone to the same solution with 10 µM InsP3, the L-type Ca2+ current simply ran down from an initial 0.89 nA (control) to 0.80 nA (in 10 µM InsP3). When the InsP3 solution was replaced with one containing 10 µM cAMP, ICa(L) increased to 1.1 nA. Upon washout of cAMP with the initial control pipette solution, ICa(L) decreased to 0.77 nA.


Figure 6
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  		Fig. 6 cAMP (10 µM) but not InsP3 (10 µM) increased ICa(L) in the presence of intrapipette Cs+ and EGTA. The voltage clamp protocol consisted of voltage jumps from –80 to –40 mV for 350 ms followed by a 300-ms step to +10 mV before returning to the holding potential. From left to right, the current traces are taken from control (a), at 3 min in 10 µM InsP3 (b), at 4 min in 10 µM cAMP (c), and at 6 min after washout (d). Calibrations for current, time and zero current apply to all traces.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
We tested two hypotheses pertaining to the proposed messenger function of InsP3 in heart cells. Does InsP3 increase ICa(L) by a direct action on the L-type Ca2+ channel? Does InsP3 modulate E–C coupling by an indirect action via the SR? These hypotheses arise from previous attempts to ascertain the mechanism for the stimulatory effect of ACh and CCh on mammalian ventricle. Choline ester agonists increased the contraction force in guinea pig ventricular muscle by occupancy of muscarinic receptors [2–5]. We (reviewed in [22]) and others [23]have suggested that altered Na/Ca exchange and/or increased InsP3 levels might be involved in the increased contractions caused by CCh. Thus far, the evidence does not exclude either mechanism [1]. Our new results show that InsP3 does not change the L-type Ca2+ current that evokes contractions when applied internally through the recording pipette to guinea pig ventricular myocytes. We found that InsP3 modulates contractions evoked by ICa(L) in a concentration- and time-dependent manner. Neither the pattern of the contraction effects nor the lack of effect on ICa(L) is consistent with the hypotheses that InsP3 functions as a messenger in the heart for muscarinic agonists that mimic the stimulatory effect of the parasympathetic transmitter, ACh.

4.1 InsP3 and ICa(L)
We did not detect any effect on ICa(L) of InsP3 at either low or high pipette concentrations in experiments with EGTA present, to buffer intracellular Ca2+, and with Cs+, to suppress K+ currents. These experiments were performed under conditions that led others [8]to propose the hypothesis that InsP3 increased ICa(L). The increased entry of trigger Ca2+ was assumed to account for the augmented intracellular Ca2+ transients by CCh [17]. Our results do not support the hypothesis that InsP3 is the messenger for the stimulant action of CCh on ICa(L) in guinea pig ventricular myocytes. In contrast to skeletal muscle [18], InsP3 does not appear to have a receptor on or near cardiac L-type Ca2+ channels that regulates this current. Also, InsP3 did not change ICa(L) under conditions that allowed detection of contractions via SR Ca2+ release. Neither the amplitude nor the rates of inactivation changed beyond the spontaneous run-down that occurs in such experiments (see also ref. [19]). With either K+-rich or Cs+-rich pipette solution, cAMP increased ICa(L), as expected for this messenger, which phosphorylates a protein associated with the channel (Figs. 1, 5 and 6GoGo). To our knowledge, there are no reports of the physiological concentrations of InsP3 in heart cells. The pipette concentrations we tested have been used by others [11–13, 24]and ourselves [14–16]in experiments that demonstrated the mobilization by InsP3 of SR Ca2+.

4.2 InsP3 effects on contractions
At 1–10 µM, InsP3 transiently increased contractions by 30–40%. Higher concentrations (50–300 µM) only reduced myocyte contractions reversibly. During dialysis, InsP3 did not induce contractions per se, but rather modulated contractions evoked by ICa(L). These results are consistent with the functional evidence for the presence of InsP3 receptors regulating Ca2+ release from cardiac SR (see Section 1). Furthermore, the type 1 InsP3 receptor mRNA is expressed in heart and its receptor is co-localized with the cardiac isoform of the ryanodine receptor in rat heart myocytes (reviewed in ref. [25]). While there is much evidence for the presence and function of InsP3 receptors on the SR of heart muscle, it should also be noted that InsP3, up to 200 µM, did not change myofilament calcium sensitivity [12, 14, 24].

We observed that low (3 µM) and high (100 µM) pipette concentrations of InsP3 increased and decreased, respectively, the magnitude of caffeine (10 mM)-induced contractures. The results at low concentration are like those reported in saponin-permeabilized guinea pig ventricle, where InsP3 (1–25 µM) augmented the rate of development, amplitude and duration of contractures by submaximal caffeine concentrations [12]. At maximal caffeine concentrations (25 mM), InsP3 had less effect on contracture amplitude but reduced the rate of relaxation. It was proposed that InsP3 released Ca2+ from the same SR pool as caffeine and that, like caffeine, InsP3 increased the sensitivity to Ca2+ [12].

The sustained decrease of cell shortening and of caffeine-induced contractures at higher pipette concentrations of InsP3 could arise if Ca2+ were depleted from the InsP3-sensitive SR pool. Intracellular Ca2+ transients and cell contractions diminished in rat myocytes exposed to {kappa} and {delta} opioids [26]. These results were taken to indicate SR Ca2+ depletion by InsP3, the synthesis of which was increased by these opioids. In our experiments, one might suppose that the replenishment of SR Ca2+ by Ca2+ entering through L-type channels lags behind the depletion caused by persistent activation of SR release channels through the InsP3 receptor. Alternatively, high InsP3 concentrations could have reduced the sensitivity of the release mechanism to CICR and caffeine. Our experiments were not designed to distinguish between these possibilities.

The concentration dependence for stimulation of ICa(L)-evoked contractions by InsP3 in guinea pig ventricular myocytes is quite similar to that reported for InsP3-induced contractions in mechanically skinned rat ventricle [11], saponin-permeabilized chick atrial muscle [14]and for InsP3-induced stimulation of the Na/Ca exchange current (INa/Ca) in guinea pig ventricular myocytes [15]. PPS, an InsP3 receptor antagonist [25], prevented the increased contractions by InsP3 and suppressed InsP3-induced increases in INaCa in guinea pig ventricular myocytes [15]. A sustained increase of cell contractions occurred when PPS alone was introduced into the myoplasm. This action might arise if PPS removed inhibition by endogenous InsP3. Alternatively, in addition to acting as an InsP3 receptor antagonist, PPS activates the ryanodine receptor in skeletal muscle SR [27]in a Ca2+-dependent manner [28]. On the favorable assumption that PPS has a similar action on ryanodine-sensitive Ca2+ release channels of cardiac SR, an increased extent of cardiac cell contractions is expected.

4.3 InsP3 as mediator of muscarinic agonist-induced contractile stimulation
InsP3 was suggested as a mediator of the positive inotropic effect of CCh (see Section 1). The biphasic contraction pattern at low InsP3 pipette concentrations differs from the sustained increase of contractions seen with CCh in papillary muscle [2–5]and single myocytes [6, 7]from guinea pig heart. The distinction between the InsP3 effect and that of CCh cannot be explained by a difference in stimulus frequency. The positive inotropic effect of CCh in guinea pig papillary muscle and in single myocytes is greatest at frequencies <1 Hz [2, 7].

There is another reason to question the role of InsP3 as a mediator of muscarinic stimulation of contractions. It is predicted that a substance that increases SR Ca2+ release without changing either the trigger or myofilament Ca2+ sensitivity should have only a transient stimulatory effect on contractions [29]. At low concentrations, caffeine caused only a transient increase in systolic Ca2+ and of contractions evoked by ICa(L) [30]. Our results during application of 1–10 µM InsP3 are like those seen with low concentrations of caffeine, which causes a sustained release of SR Ca2+. A difference between caffeine and InsP3 is the lack of a transient reduction of contractions upon washout of InsP3. The pipette dialysis method does not change solution composition as quickly as do the superfusion devices used with external caffeine application. Slow washout of the dialysate could account for the failure to detect a transient reduction in contractions after removal of low concentrations of InsP3. When high InsP3 concentrations, which reduced contractions, were removed, some 3 min elapsed before recovery. The details of the model indicate that agents whose only action is to increase or decrease the rate of SR Ca2+ release will have only transient stimulatory or inhibitory effects, respectively [29]. In this regard, previous examinations of the mechanism of muscarinic stimulation led to the conclusion that CCh increases cell contractions by augmenting SR Ca2+ content [6, 7]. Suffice to state that ACh or CCh increases intracellular Na [2, 4]by promoting its entry through tetrodotoxin-resistant channels [31]. The increased intracellular Na+, by altering Na/Ca exchange, eventually results in an increased SR Ca2+ content [6]. As predicted by the model [29], the increased SR Ca2+ content allows CCh to cause a sustained increase of Ca2+ transients and contractions; ICa(L) was not changed [6, 7].

Carbachol should also increase the other messenger of the phosphoinositide cascade, diacylglycerol (DAG). While this activator of protein kinase C (PKC) could serve to increase ICa(L), divergent results have been obtained by many investigators (reviewed in [32]). The phorbol ester, TPA (12-O-tetradecanoylphorbol-13-acetate), which activates PKC, did not enhance ICa(L) [19]. Also, PKC activation by phorbol ester produced negative inotropic effects in guinea pig papillary muscle and chick atria, and suppressed the positive inotropic action of CCh [4, 5, 33]. When a DAG analogue was photolytically released within the myoplasm, a sustained positive inotropic action occurred, while a negative inotropic effect was produced by external addition [34]. The contrasting results between intracellular and extracellular application of the DAG analogue provide a note of caution in the interpretation of results.

4.4 Limitations
The effects we observed are at variance with the hypotheses that InsP3 is a mediator of the stimulation of contractions or ICa(L) by muscarinic agonists. Several considerations temper this conclusion.

We cannot exclude the possibility of differential metabolism of dialyzed InsP3 compared to that of endogenously synthesized InsP3. Cytoskeleton-associated aldolase binds InsP3 and functions as a sink for this messenger in porcine tracheal smooth muscle [35]. However, the fact that contractions ‘rebounded’ upon removal of lower InsP3 concentrations militates against metabolism as a mechanism for the transient nature of increased myocyte shortening during dialysis. The biphasic effect of myoplasmic Ca2+ on InsP3-induced contractions [16], together with the action of InsP3 restricted to the SR, could explain the transient stimulation of contractions at lower InsP3 concentrations. We previously found that the potentiation of CICR by InsP3 in SR of avian heart muscle was also transient [16].

Variations in intracellular Ca2+ may facilitate or inhibit ICa(L) (reviewed in ref. [32]). The release of Ca2+ by dialyzed InsP3 may be too slow to raise myoplasmic Ca2+ above threshold concentrations at the inner mouth of L-type Ca2+ channels. Thus, one might speculate that InsP3-activated SR Ca2+ release channels, while co-localized with ryanodine-sensitive Ca2+ release channels [25], do not display a preferential disposition with L-type Ca2+ channels as do ryanodine-sensitive Ca2+ release channels [36].

Within these limitations, the available evidence for Ca2+ mobilization by InsP3, either at plasma membrane L-type Ca2+ channels or at SR Ca2+ release channels, appears insufficient to support its function as a messenger for the cardiostimulatory effects of muscarinic agonist.

Time for primary review 24 days.


   Acknowledgements
 
This work was supported by USPHS Grant HL-13339. We thank Ms. René Bumbera for help in preparing the manuscript.


   Notes
 
1 Present address: Third Department of Internal Medicine, Nagoya City University Medical School, 1 Kawasumi Mizuko-cho, Mizuko-ku, Nagoya 467, Japan. Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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